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PubMed1992

A histochemical study of acid phosphatases in medullary bone matrix and osteoclasts in laying Japanese quail.

Acid phosphatase activity in medullary bone matrix and osteoclasts of laying Japanese quail was examined histochemically. To avoid nonspecific staining, the reactivity of the enzyme was evaluated using both the azo dye method and the lead salt method and nonembedded thick sections and resin-embedded thin sections. The pH of the incubation medium was also varied from the acid range (pH 5.0 and 6.5) to the alkaline range (pH 8.5). Medullary bone osteoclasts contain both tartrate-resistant acid phosphatase (TRAP) activity and fluoride-resistant acid phosphatase (FRAP) activity, and no significant difference in intensity was detected between active and inactive osteoclasts. The entire matrix of medullary bone was positive for tartrate-resistant, fluoride-sensitive acid phosphatase activity. No reaction product was observed in sections incubated in substrate-free and pH 8.5 media. The results demonstrate the existence of FRAP in medullary bone osteoclasts and suggest that medullary bone matrix includes TRAP throughout the matrix.

Acid Phosphatase↗

PubMed1999

Augmentation of the nasal spine area with tissue bone matrix sponge.

OBJECTIVE: High-density tissue bone matrix (TBM) sponge is a homograft derived from human cadaver. It is reported to be osteoinductive. The objective of this paper is to measure the bone formation and cosmetic effect of TBM sponge implanted in the nasal spine area of patients undergoing rhinoplasty with retraction of this region and loss of nasal tip support. DESIGN: The study was designed as a prospective trial. SETTING: Patients were selected from private facial cosmetic practice and from public otolaryngology practice. PATIENTS: Six patients were selected who had retraction of the nasal spine area and loss of tip support. INTERVENTION: Patients had implantation of the TBM sponge in the nasal spine area either as a sole procedure or in conjunction with other rhinoplasty maneuvers. MAIN OUTCOME MEASURES: Palpation was used to assess position, size and consistency of the implant. Cosmetic effect was assessed by computer imaging, which was used to measure nasolabial angle and tip projection. Bone formation was assessed by computed tomography scanning. RESULTS: The implant could be palpated in all six patients at 1 month postoperatively, but at 3 months was either smaller or could not be felt. Nasolabial angle and tip projection were improved in all patients initially, but 3 months following surgery this cosmetic improvement was maintained in only two patients. At 3 months, CT scanning showed no evidence of bone formation. CONCLUSION: The TBM sponge was not found to be osteoinductive or permanent when implanted in the nasal spine area and therefore is not a good implant in that region.

Bone Matrix↗

PubMed2003

Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix.

A sensitive, rapid, reliable and quantitative method to check the bone forming potential of demineralized bone matrix (DBM) has been developed. The osteoinductivity of the bone morphogenetic proteins (BMPs), present in DBM, can be measured in vitro using a pluripotent myoblast C2C12 cell line. Alkaline phosphatase activity induced by co-incubation of DBM with C2C12 cells was dose-responsive and corresponds to the amount of active BMPs in DBM. Bone forming potential was simultaneously tested in vivo by implanting DBM intra-muscularly in nude rats. ALP activity induced in C2C12 cells, correlated with bone formation in vivo (r=0.88), determined by alkaline phosphatase activity, mineralization density and histomorphology of the DBM explants. Results from DBM batches, originating from five established Bone Banks, showed good consistency between in vitro and in vivo assays. However, DBM activity varied widely from bank to bank as well as from batch to batch within the same bank.

Alkaline Phosphatase↗

PubMed1988

Polypeptide growth factors in bone matrix.

The presence of many types of polypeptide growth factors in the mineralized extracellular matrix of bone is now well established. These factors are generally referred to as bone-derived growth factors (BDGFs), and are similar, or possibly identical, to the following species; platelet-derived growth factor (PDGF); acidic and basic forms of fibroblast growth factor (aFGF, bFGF); transforming growth factor beta (TGF-beta); and insulin-like growth factor 1 (IGF-1). Several osteoinductive factors, such as bone morphogenetic protein (BMP) and osteogenin, a skeletal growth factor (SGF), and osteoblast-derived BDGFs, have also been identified. Complete description of the biological functions of these BDGFs which are relevant to bone will ultimately require specific bioassays involving specific cell types in vitro, as well as in vivo animal implant models. Studies with primary rat osteoblast-like cells exposed either to mixed BDGFs, pure TGF-beta, or heparin-purified PDGF, aFGF, or bFGF from bovine bone have shown a general dose-dependent mitogenic effect. Phenotypic changes which accompany the BDGF-induced wave of proliferation include: decreased osteocalcin secretion and a reduction in 1,25-(OH)2 vitamin D3-stimulated osteocalcin synthesis; reduced alkaline phosphatase specific activity; decreased cyclic AMP responsiveness to parathyroid hormone (PTH); and increased collagen synthesis. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than that in serum. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for the unmasking or release of BDGFs from the mineralized matrix that result in local action on osteoblasts, endothelial cells, and other target cells are undoubtedly important for the development and maintenance of bone tissue.

Animals↗

PubMed1989

In vivo analysis of the half-life of the osteoinductive potential of demineralized bone matrix using diffusion chambers.

Subcutaneous implantation of demineralized bone matrix (DBM) from rat initiates a sequence of developmental events that results in endochondral bone formation. This investigation examined the modification of the osteoinductive potential of DBM during the initial stages of this developmental cascade. Diffusion chambers (DC), constructed with filters of known pore size, permitting or excluding cells from entering the chambers, and containing DBM were subcutaneously implanted into Long-Evans male rats for specific time periods (1-7 days). DC were recovered and the osteoinductive potential of the matrix from these chambers was then tested by subcutaneous implantation and assaying the resulting day 11 plaque tissue enzymatically for alkaline phosphatase activity, and histologically for evidence of chondrogenesis and osteogenesis. The possible modification of DBM by local systemic factors (enzymatic degradation) or contact by polymorphonuclear leukocytes (PMNs) was also investigated. We have concluded from this study that the osteoinductive potential of DBM has a half-life of 5-7 days following implantation and although the enzymes collagenase, elastase, and trypsin abolished this activity, pepsin significantly enhanced it. Culture of PMNs with matrix prior to its implantation appeared to have little effect. Furthermore, during the initial stages of matrix-induced endochondral bone formation, DBM serves as both the instructive inducer and permissive substratum required in this process.

Alkaline Phosphatase↗

PubMed2005

[An experimental study on the influence of osteoporosis to bone repairing with bone matrix gelatin in ovariectomized rats].

OBJECTIVE: To study the influence of experimentally osteoporosis to osteogenic efficiency of bone matrix gelatin(BMG) implanted into the calvarial defects of rats. METHODS: Sixty-eight female SD rats of 12 +/- 1 weeks were randomly divided into two groups with 34 rats in each group. The ovaries were excised in the ovariectomized group (VG). The control group underwent sham surgery. Ninety days after ovariectomy, 10 rats from each group were examined to ensure the formation of postmenopausal osteoporosis by measuring bone density of the femur with single photon absorptiometric measurements. A critical-sized (8 mm in diameter) calvarial defect was created on the rest of 48 rats. Bone matrix gelatin was implanted to the defect. The rats were scarified at the 21st and 56th day after surgery respectively. The new bone forming capability of BMG was evaluated with undecalcified histological observation, tetracycline fluorescence marker, quantitative bone histomorphometry, At 90th day after ovariectomy, bone density of scanning electron microscopy and X-ray spectrometry. RESULTS: OVG showed very significant difference compared with the control group (0.315 +/-.015) g/cm2 vs [(0.347 +/- 0.017) g/cm2, P < 0.01 ]. At the 21st day following the implantation operation, new bone formed within the bone defects in both groups. The amount of new bone in OVG was lower than the control group. The tetracycline-labeled region in the bone defect was sparser in the OVG. At the 56th day, the bone defects healed mostly in the control group but fibrous tissue filled parts of bone defect in the OVG. The distance between two fluorescent lines of incorporated tetracycline and the mean mineralization deposition were significantly lower in the OVG than the control at the 21st day and 56th day. Mineralization of callus in OVG was inferior. Significant difference was found between the OVG and the control group in the calcium to phosphate ratio of callus in bone defects at the two time-points. CONCLUSIONS: Experimentally induced osteoporosis depressed osteogenic efficiency of BMG, suggesting that estrogen could play an important role in bone remodeling with bone substitute participating.

Animals↗

PubMed1993

Degradation of bone matrix proteins by osteoclast cathepsins.

1. The degradation of the bone matrix proteins osteocalcin, osteonectin and alpha 2HS-glycoprotein by human cathepsins B and L and human osteoclastoma cathepsins has been investigated. 2. Intermediate degradation products (M(r) > 12 kDa) were not observed during the digestion of alpha 2HS-glycoprotein and osteonectin by cathepsins B and L although they were observed with some of the osteoclastoma cathepsins. Most of the osteoclastoma cathepsins were capable of degrading these two proteins to small peptides at comparable rates. 3. Each cathepsin produced a different pattern of osteocalcin degradation products. 4. The extensive range of non-collagenous proteins in bone matrix may necessitate the production by osteoclasts of cathepsins with different specificities during bone resorption.

Blood Proteins↗

PubMed1984

Enhancement of bone ingrowth by the use of bone matrix as a biologic cement.

In an effort to enhance bone growth into implants with porous coatings, a decalcified bone cement was used to secure fixation of the implants in canine femora. Rods with porous coatings were inserted bilaterally into the femoral medullary canals of nine dogs of varying sizes. A demineralized bone paste was used to enhance the fixation in one side, with the contralateral limb serving as a control. Pull-out testing and gross and roentgenographic examination of the specimens confirmed that the demineralized bone matrix enhanced fixation.

Animals↗

PubMed1986

Osteogenesis induced by bone matrix is inhibited by inflammation.

This study was designed to examine the effect of inflammatory reaction elicited by percutaneous tube on bone induction. Inflammation was provoked by different types of biomaterials. In order to evaluate incorporation of percutaneous tubes, bone matrix and subcutaneous tissue, demineralizing bone matrix was implanted in the subcutaneous tissue of rats and was exposed to interaction with inflammatory conditions. Inhibition to the induction of cartilage and bone by the inflammatory process could be clearly demonstrated. It is suggested that the low pH levels, typical to enzymes operative in inflammation are a direct cause for inhibition of chondro and osteogenesis. The process of calcification is characterized by the activation of enzymes in high pH levels.

Animals↗

PubMed1991

In vitro chemotactic response of osteoblast-like osteosarcoma cells to a partially purified protein extract of demineralized bone matrix.

The purpose of this investigation was to evaluate the chemotactic potential of a partially purified protein extract from bone matrix when tested against osteosarcoma cells with osteoblast characteristics. The chemotactic response of ROS 17/2 cells to a lyophilized bovine bone extract purified to "Urist step eight" was evaluated in Boyden blind well chambers. A checkerboard design was employed to test cell migration against positive, negative, and no concentration gradients, thereby controlling the effects of chemokinesis and/or random migration on results. The results demonstrate that the partially purified protein extract from bovine bone matrix is chemotactic since more cells migrated to positive gradients than to negative gradients (P less than .01). The chemotactic effect was confirmed by an increase in cell migration toward positive gradients of the bone extract compared to cell migration in the presence of no gradient (P less than .01). When no gradient was present, the cells exhibited an increased response in the presence of equal concentrations of the bone extract (P less than .01) indicating a chemokinetic effect. The proteinaceous nature of the chemoattractant was confirmed by its susceptibility to trypsin digestion and heat exposure.

Animals↗

PubMed2004

Influence of fluor salts, hormone replacement therapy and calcitonin on the concentration of insulin-like growth factor (IGF)-I, IGF-II and transforming growth factor-beta 1 in human iliac crest bone matrix from patients with primary osteoporosis.

OBJECTIVE: Data from cell culture experiments suggest that local growth factors (GFs) may mediate the effects of estrogens, calcitonin or fluor ions on the skeleton. To assess the in vivo relevance of the in vitro reports, the effect of fluor salts, hormone replacement therapy (HRT) and calcitonin on the concentrations of IGF-I, IGF-II and transforming growth factor (TGF)-beta 1 in bone matrix extracts from osteoporotic patients was evaluated. DESIGN: Iliac crest bone biopsies were obtained from 170 patients (76 men and 94 women) with primary osteoporosis aged 55.5+/-0.8 Years. METHODS: Bone matrix extraction was performed based on a guanidine-HCl/ethylendiamine-tetra-acetic acid method. RESULTS: In comparison with age- and body mass index (BMI)-matched controls, no influence of long-term therapy with fluor ions (n=41) or calcitonin (n=16) on the bone matrix concentration of GFs was noticed. Postmenopausal women with osteoporosis on HRT (n=39) had lower skeletal IGF-I but not IGF-II levels as compared with age- and BMI-matched non-users. However, the lower rate of bone turnover in women with HRT may account for this difference, since the significance was lost after adjustment for alkaline phosphatase. Likewise, a tendency for lower TGF-beta 1 levels was observed in HRT users as compared with non-users but was lost after adjustment for bone turnover. None of the therapies influenced the serum levels of GFs when patients receiving continuous therapy for at least 1 Year before bone biopsy were considered. CONCLUSIONS: Our data suggest no direct effect of fluor therapy on skeletal GFs levels. At the concentrations used, neither HRT nor calcitonin appeared to exert any significant influence on serum or bone matrix GF levels.

Biopsy↗

PubMed1998

Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus?

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual.

Acid Phosphatase↗

PubMedDate not supplied

Bone matrix insulin-like growth factor (IGF)-I, IGF-II and transforming growth factor (TGF)-beta1 levels in men and postmenopausal women with osteoporosis: lack of association with circulating growth factors and bone mineral density.

Previous clinical studies have suggested a positive correlation between serum insulin-like growth factor components and bone mass in both men and women with or without osteoporosis. The aim of the present study was to analyze the relationship between the skeletal levels of insulin-like growth factors and transforming growth factor-b1 and bone mineral density in a group of men and postmenopausal women in whom osteoporosis was diagnosed previously. Bone matrix extraction was achieved by passive dialysis against tetrasodium EDTA-guanidine-HCL. IGF's were quantified by radioimmunoassay. TGF-b1 was assessed by a specific enzyme-linked immunoassay. No correlation between BMD and the concentration of IGF-I, IGF-II and TGF-b1 in bone matrix was detected in either men or postmenopausal women with osteoporosis. In addition, circulating growth factors levels failed to be associated with the concentration of IGF-I, IGF-II and TGF-b1 in the skeleton. Thus, our study provides no evidence for a major role of bone matrix IGF's or TGF-b1 as determinants of bone mass in men or postmenopausal women with osteoporosis.

Algorithms↗

PubMed2004

Retroviral delivery of Noggin inhibits the formation of heterotopic ossification induced by BMP-4, demineralized bone matrix, and trauma in an animal model.

BACKGROUND: The heterotopic ossification of muscles, tendons, and ligaments is a common problem faced by orthopaedic surgeons. We investigated the ability of Noggin (a BMP [bone morphogenetic protein] antagonist) to inhibit heterotopic ossification. METHODS: Part 1: A retroviral vector carrying the gene encoding human Noggin was developed and used to transduce muscle-derived stem cells. Part 2: Cells transduced with BMP-4 were implanted into both hind limbs of mice along with either an equal number, twice the number, or three times the number of Noggin-expressing muscle-derived stem cells (treated limb) or with nontransduced muscle-derived stem cells (control limb). At four weeks, the mice were killed and radiographs were made to look for evidence of heterotopic ossification. Part 3: Eighty milligrams of human demineralized bone matrix was implanted into the hind limbs of SCID (severe combined immunodeficiency strain) mice along with 100,000, 500,000, or 1,000,000 Noggin-expressing muscle-derived stem cells (treated limbs) or nontransduced muscle-derived stem cells (control limbs). At eight weeks, the mice were killed and radiographs were made. Part 4: Immunocompetent mice underwent bilateral Achilles tenotomy along with the implantation of 1,000,000 Noggin-expressing muscle-derived stem cells (treated limbs) or nontransduced muscle-derived stem cells (control limbs). At ten weeks, the mice were killed and radiographs were made. RESULTS: Part 1: An in vitro BMP inhibition assay demonstrated that Noggin was expressed by muscle-derived stem cells at a level of 280 ng per million cells per twenty-four hours. Part 2: Three varying doses of Noggin-expressing muscle-derived stem cells inhibited the heterotopic ossification elicited by BMP-4-expressing muscle-derived stem cells. Heterotopic ossification was reduced in a dose-dependent manner by 53%, 74%, and 99%, respectively (p < 0.05). Part 3: Each of three varying doses of Noggin-expressing muscle-derived stem cells significantly inhibited the heterotopic ossification elicited by demineralized bone matrix. Heterotopic ossification was reduced by 91%, 99%, and 99%, respectively (p < 0.05). Part 4: All eleven animals that underwent Achilles tenotomy developed heterotopic ossification at the site of the injury in the control limbs. In contrast, the limbs treated with the Noggin-expressing muscle-derived stem cells had a reduction in the formation of heterotopic ossification of 83% and eight of the eleven animals had no radiographic evidence of heterotopic ossification (p < 0.05). CONCLUSIONS: The delivery of Noggin mediated by muscle-derived stem cells can inhibit heterotopic ossification caused by BMP-4, demineralized bone matrix, and trauma in an animal model. CLINICAL RELEVANCE: Gene therapy to deliver Noggin may become a powerful method to inhibit heterotopic ossification in targeted areas of the body.

Achilles Tendon↗

PubMed1999

A comparative study of fibrous dysplasia and osteofibrous dysplasia with regard to expressions of c-fos and c-jun products and bone matrix proteins: a clinicopathologic review and immunohistochemical study of c-fos, c-jun, type I collagen, osteonectin, osteopontin, and osteocalcin.

Fibrous dysplasia and osteofibrous dysplasia are both benign fibro-osseous lesions of the bone and are generally seen during childhood or adolescence. Histologically, the features of these bone lesions sometimes look quite similar, but their precise nature remains controversial. We retrospectively studied clinicopathologic findings in 62 cases of fibrous dysplasia and 20 cases of osteofibrous dysplasia with regard to their anatomic location and histological appearance. From among these cases, the immunohistochemical expressions of c-fos and c-jun proto-oncogene products and bone matrix proteins of type I collagen, osteonectin, osteopontin, and osteocalcin were evaluated in 20 typical fibrous dysplasias and 17 osteofibrous dysplasias using paraffin sections, and these expressions were then assessed semiquantitatively. Microscopically, fibrous dysplasia showed various secondary changes, such as hyalinization, hemorrhage, xanthomatous reaction, and cystic change in 22 of the 62 cases (35%). This was a higher incidence than in osteofibrous dysplasia, in which only 2 of the 20 cases (10%) showed such changes. In the elderly fibrous dysplasia cases, the cellularity of fibroblast-like cells was rather low, and those cases were hyalinized. Almost all of the cases of fibrous dysplasia and osteofibrous dysplasia showed positive expressions of c-fos and c-jun products. The expressions of type I collagen and osteopontin showed no difference between fibrous dysplasia and osteofibrous dysplasia. Immunoreactivity for osteonectin in bone matrix was detected in only 1 case of fibrous dysplasia (1 of 20), whereas it was recognized in 14 of the 17 cases of osteofibrous dysplasia. Furthermore, the immunoreactivity for osteocalcin in bone matrix and fibroblast-like cells was higher in fibrous dysplasia than it was in osteofibrous dysplasia, semiquantitatively. Our immunohistochemical results regarding osteonectin and osteocalcin suggest that the bone matrix of fibrous dysplasia is somewhat more mature than that of osteofibrous dysplasia, and that the fibroblast-like cells in fibrous dysplasia share some phenotypic features with osteoprogenitor cells of normal osteogenic tissues. Fibrous dysplasia and osteofibrous dysplasia share some similar histological features, including c-fos and c-jun expressions, although different clinicohistologic features and immunohistochemical expressions of osteonectin and osteocalcin were observed. These features suggest that the mechanisms behind the development of fibrous dysplasia and osteofibrous dysplasia are similar, but this is not necessarily indicative of a closer relationship between the 2 diseases.

Adolescent↗

PubMed2002

Efficacy of contained metaphyseal and periarticular defects treated with two different demineralized bone matrix allografts.

The efficacies of two different allografts, Grafton (demineralized bone matrix [DBM] in a glycerol carrier) and Orthoblast (DBM in a reverse thermal poloxamer carrier) were examined from cases involving periarticular fractures. Demographic, perioperative, and outcome data for patients with periarticular fractures who underwent a prospectively designed protocol for bone grafting were compiled, with 15 cases using Orthoblast and 13 using Grafton. A successful graft was defined as healing on the first graft attempt without complications. Healing was determined by radiographic studies and clinical evaluation. The successful graft rates of Orthoblast and Grafton were 15/15 and 9/13, respectively.

Adult↗

PubMed1987

Altered organization of non-collagenous bone matrix in osteoporosis.

In osteoporosis it is postulated that while the amount of bone is diminished, the quality of the bone is unaltered. Recently relatively novel methods of analysis have shown that, at the fracture site of osteoporotic subcapital fractures, there is a marked change in the molecular orientation of components of the non-collagenous bone matrix. These procedures, now applied to iliac crest biopsies, confirm earlier findings of altered orientation of the proteoglycans at the subcapital fracture site but show that very similar changes occur even in the iliac crests from patients with both types of osteoporotic proximal femoral fracture. Thus, whereas the amount of these acidic moieties of the non-collagenous bone matrix was unchanged, the molecular orientation was markedly altered, albeit not to the same extent as that found at the fracture site. These results imply that the quality of the bone, as well as the quantity, may be generally affected in osteoporosis.

Adult↗

PubMed2002

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.

Animals↗